Project summary NKT cells are a conserved population of CD Id-restricted innate-like lymphocytes with a hybrid NK/T phenotype. They critically regulate a broad range of disease conditions by releasing Th1 or Th2 cytokines and chemokines upon interactions with DC, macrophages or B cells. The dual specificity of their semi- invariant Va14-Ja18 (Va24-Ja18 in humans) TCR for the self glycosphingolipid iGb3 and for microbial a- glycosylceramides underlies important roles in infectious diseases. In particular NKT cells are essential for defense against alpha-proteobacteria, including some Rickettsiales that are recognized agents of emerging infectious diseases. NKT cells also exert immunoregulation of some primary or transplanted cancers and regulate various autoimmune and inflammatory diseases. The overarching goal of this project is to understand the developmental steps of the NKT cell lineage and the mechanisms underlying its commitment and differentiation. Our previous work identified the self glycosphingolipid iGb3 as a natural ligand of NKT cells and demonstrated the critical importance of early interactions between NKT thymic precursors and CDId-expressing cortical CD4+CD8+ thymocytes. The specific aims will test the hypothesis that !Gb3 is the thymic ligand driving selection by measuring iGb3 expression in the thymus and defining the iGb3-specific TCR Vp repertoire (SA#1), develop a conditional iGb3 synthase mutant mouse (SA#2), test the role of iGb3 and other ligands at different stages of NKT cell development (SA#3) and test the hypothesis that SLAM family members provide the elusive second signal required for NKT development (SA#4). Relevance NKT lymphocytes represent an essential component of immunity against pathogens associated with emerging infectious diseases, such as Rickettsiales. There are marked individual variations in NKT cell frequency and function in the human population and in individual mouse strains. Thus, investigating the basic mechanisms underlying NKT cell development may be important not only to enhance our general knowledge of lymphocyte development, but also for understanding susceptibility to some of the emerging infectious diseases.